Polypeptides having peroxygenase activity

ABSTRACT

The present invention relates to isolated polypeptides having peroxygenase activity, and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides. A polynucleotide encoding a peroxygenase was isolated from  Myceliophthora fergusii.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/EP2013/070963 filed Oct. 8, 2013, which claims priority or thebenefit under 35 U.S.C. 119 of European application no. 12188453.0 filedOct. 12, 2012 and U.S. provisional application No. 61/716,133 filed Oct.19, 2012. The content of each application is fully incorporated hereinby reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND

Field of the Invention

The present invention relates to polypeptides having peroxygenaseactivity, and polynucleotides encoding the polypeptides. The inventionalso relates to nucleic acid constructs, vectors, and host cellscomprising the polynucleotides as well as methods of producing and usingthe polypeptides.

Description of the Related Art WO 2006/034702 A1 discloses methods forthe enzymatic hydroxylation of non-activated hydrocarbons, such as,naphtalene, toluoyl and cyclohexane, using the AaP peroxygenase enzymeof Agrocybe aegerita TM A1. This is also described in Ullrich andHofrichter, 2005, FEBS Letters 579: 6247-6250.

DE 103 32 065 A1 discloses methods for the enzymatic preparation ofacids from alcohols through the intermediary formation of aldehydes byusing the AaP peroxygenase enzyme of Agrocybe aegerita TM A1.

A method was reported for the rapid and selective spectrophotometricdirect detection of aromatic hydroxylation by the AaP peroxygenase(Kluge et al., 2007, Appl Microbiol Biotechnol 75: 1473-1478).

Another peroxygenase capable of aromatic peroxygenation was isolatedfrom the coprophilous fungus Coprinus radians and characterized, theN-terminal 16 amino acids were identified and aligned with theN-terminal 14 amino acids of the AaP enzyme of the A. aegerita strainearlier published; but the encoding gene was not isolated (Anh et al.,2007, Appl Env Microbiol 73(17): 5477-5485).

WO 2008/119780 discloses several different peroxygenase polypeptides andtheir encoding polynucleotides, as well as recombinant productionthereof.

WO 2011/120938 discloses site-specific hydroxylation of aliphatichydrocarbons using peroxygenase polypeptides.

The present invention provides novel polypeptides having peroxygenaseactivity and polynucleotides encoding the polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides havingperoxygenase activity selected from the group consisting of:

-   (a) a polypeptide having at least 60% sequence identity to the    mature polypeptide of SEQ ID NO: 2;-   (b) a polypeptide encoded by a polynucleotide that hybridizes under    medium stringency conditions with (i) the mature polypeptide coding    sequence of SEQ ID NO: 1, (ii) the cDNA sequence thereof, or (iii)    the full-length complement of (i) or (ii);-   (c) a polypeptide encoded by a polynucleotide having at least 60%    sequence identity to the mature polypeptide coding sequence of SEQ    ID NO: 1, or the cDNA sequence thereof;-   (d) a variant of the mature polypeptide of SEQ ID NO: 2 comprising a    substitution, deletion, and/or insertion at one or more (e.g.,    several) positions; and-   (e) a fragment of the polypeptide of (a), (b), (c), or (d) that has    peroxygenase activity.

The present invention also relates to isolated polynucleotides encodingthe polypeptides of the present invention; nucleic acid constructs;recombinant expression vectors; recombinant host cells comprising thepolynucleotides; and methods of producing the polypeptides.

The present invention also relates to methods of using the polypeptidesof the invention.

The present invention also relates to a polynucleotide encoding a signalpeptide comprising or consisting of amino acids −17 to −1 of SEQ ID NO:2, which is operably linked to a gene encoding a protein; nucleic acidconstructs, expression vectors, and recombinant host cells comprisingthe polynucleotides; and methods of producing a protein.

DEFINITIONS

Peroxygenase: The term “peroxygenase” means an “unspecific peroxygenase”activity according to EC 1.11.2.1, that catalyzes insertion of an oxygenatom from H₂O₂ into a variety of substrates, such as nitrobenzodioxole.For purposes of the present invention, peroxygenase activity isdetermined according to the procedure described in Example 2, or in M.Poraj-Kobielska, M. Kinne, R. Ullrich, K. Scheibner, M. Hofrichter, “Aspectrophotometric assay for the detection of fungal peroxygenases”,Analytical Biochemistry (2012), vol. 421, issue 1, pp. 327-329.

In one aspect, the polypeptides of the present invention (peroxygenases)have at least 20%, e.g., at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, or at least 100% ofthe peroxygenase activity of the mature polypeptide of SEQ ID NO: 2.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Fragment: The term “fragment” means a polypeptide or a catalytic domainhaving one or more (e.g., several) amino acids absent from the aminoand/or carboxyl terminus of a mature polypeptide or domain; wherein thefragment has peroxygenase activity. In one aspect, a fragment containsat least 240 amino acid residues (e.g., amino acids 1 to 240 of SEQ IDNO: 2, or amino acids 20 to 240 of SEQ ID NO: 2), at least 230 aminoacid residues (e.g., amino acids 1 to 230 of SEQ ID NO: 2, or aminoacids 20 to 230 of SEQ ID NO: 2), or at least 220 amino acid residues(e.g., amino acids 1 to 220 of SEQ ID NO: 2, or amino acids 20 to 220 ofSEQ ID NO: 2).

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Anisolated substance may be present in a fermentation broth sample.

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 1 to 244 of SEQ ID NO: 2, based on theSignalP 3.0 program, that predicts amino acids −17 to −1 of SEQ ID NO: 2is a signal peptide. It is known in the art that a host cell may producea mixture of two of more different mature polypeptides (i.e., with adifferent C-terminal and/or N-terminal amino acid) expressed by the samepolynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving peroxygenase activity. In one aspect, the mature polypeptidecoding sequence is nucleotides 52 to 88, 178 to 415, 535 to 806, and 923to 1107 of SEQ ID NO: 1, or the cDNA sequence thereof, based on theSignalP 3.0 program that predicts nucleotides 1 to 51 of SEQ ID NO: 1encode a signal peptide.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5× SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and either 35%formamide, following standard Southern blotting procedures for 12 to 24hours. The carrier material is finally washed three times each for 15minutes using 2×SSC, 0.2% SDS at 60° C.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the—nobrief option) is usedas the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

In an embodiment, the length of the alignment is at least 150 amino acidresidues, preferably at least 180 amino acid residues, more preferablyat least 200 amino acid residues, and most preferably at least 220 aminoacid residues.

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the—nobrief option) is used as the percentidentity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having peroxygenase activity. In one aspect, a subsequencecontains at least 600 nucleotides, at least 650 nucleotides, or at least700 nucleotides.

Variant: The term “variant” means a polypeptide having peroxygenaseactivity comprising an alteration, i.e., a substitution, insertion,and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5× SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5× SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 45° C.

DETAILED DESCRIPTION

Polypeptides Having Peroxygenase Activity

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 2 ofat least 60%, e.g., at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%, which have peroxygenase activity. In oneaspect, the polypeptides differ by no more than 10 amino acids, e.g., 1,2, 3, 4, 5, 6, 7, 8, or 9, from the mature polypeptide of SEQ ID NO: 2.

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 2, or an allelic variantthereof; or is a fragment thereof having peroxygenase activity. Inanother aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 2. In another aspect, the polypeptidecomprises or consists of amino acids −17 to 244 of SEQ ID NO: 2, oramino acids 1 to 244 of SEQ ID NO: 2

The polypeptides of the invention may comprise the amino acid sequenceshown as:

(SEQ ID NO: 3) Glu-His-Asp-Gly-Ser-Leu-Ser-Arg, (SEQ ID NO: 4)Glu-His-Asp-Ala-Ser-Leu-Ser-Arg, (SEQ ID NO: 5)Glu-His-Asp-Gly-Ser-Ile-Ser-Arg, or (SEQ ID NO: 6)Glu-His-Asp-Ala-Ser-Ile-Ser-Arg.which corresponds to amino acids 86-93 of SEQ ID NO: 2, and whichcoordinates a Mn atom next to a heme group.

Thus, the amino acid sequence of the polypeptides of the invention maycomprise the motif shown as (which is the result of combining SEQ ID NO:3 to SEQ ID NO: 6):

E-H-D-[G,A]-S-[L,I]-S-R. (SEQ ID NO: 7)

The polypeptides of the invention may comprise the amino acid sequenceshown as:

(SEQ ID NO: 8) Arg-Gly-Pro-Cys-Pro-Xaa-Met-Asn-Ser-Leu, (SEQ ID NO: 9)Arg-Ala-Pro-Cys-Pro-Xaa-Met-Asn-Ser-Leu, (SEQ ID NO: 10)Arg-Gly-Pro-Cys-Pro-Xaa-Leu-Asn-Ser-Leu, (SEQ ID NO: 11)Arg-Ala-Pro-Cys-Pro-Xaa-Leu-Asn-Ser-Leu, (SEQ ID NO: 12)Arg-Gly-Pro-Cys-Pro-Xaa-Met-Asn-Thr-Leu, (SEQ ID NO: 13)Arg-Ala-Pro-Cys-Pro-Xaa-Met-Asn-Thr-Leu, (SEQ ID NO: 14)Arg-Gly-Pro-Cys-Pro-Xaa-Leu-Asn-Thr-Leu, or (SEQ ID NO: 15)Arg-Ala-Pro-Cys-Pro-Xaa-Leu-Asn-Thr-Leu;which corresponds to amino acids 14-23 of SEQ ID NO: 2, and which formessential structural elements linked to the heme group. Xaa can be anyamino acid, but preferably it is Met, Gly, Ala, or Val.

Thus, the amino acid sequence of the polypeptides of the invention maycomprise the motif shown as (which is the result of combining SEQ ID NO:8 to SEQ ID NO: 15):

(SEQ ID NO: 16) R-[G,A]-P-C-P-X-[M,L]-N-[S,T]-L; and preferably(SEQ ID NO: 16) R-[G,A]-P-C-P-[M,G,A,V]-[M,L]-N-[S,T]-L.

In another embodiment, the present invention relates to an isolatedpolypeptide having peroxygenase activity encoded by a polynucleotidethat hybridizes under very low stringency conditions, low stringencyconditions, medium stringency conditions, medium-high stringencyconditions, high stringency conditions, or very high stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the cDNA sequence thereof, or (iii) the full-length complementof (i) or (ii) (Sambrook et al., 1989, Molecular Cloning, A LaboratoryManual, 2d edition, Cold Spring Harbor, N.Y.).

The polynucleotide of SEQ ID NO: 1, or a subsequence thereof, as well asthe polypeptide of SEQ ID NO: 2, or a fragment thereof, may be used todesign nucleic acid probes to identify and clone DNA encodingpolypeptides having peroxygenase activity from strains of differentgenera or species according to methods well known in the art. Inparticular, such probes can be used for hybridization with the genomicDNA or cDNA of a cell of interest, following standard Southern blottingprocedures, in order to identify and isolate the corresponding genetherein. Such probes can be considerably shorter than the entiresequence, but should be at least 15, e.g., at least 25, at least 35, orat least 70 nucleotides in length. Preferably, the nucleic acid probe isat least 100 nucleotides in length, e.g., at least 200 nucleotides, atleast 300 nucleotides, at least 400 nucleotides, at least 500nucleotides, at least 600 nucleotides, at least 700 nucleotides, atleast 800 nucleotides, or at least 900 nucleotides in length. Both DNAand RNA probes can be used. The probes are typically labeled fordetecting the corresponding gene (for example, with ³²P, ³H, ³⁵S,biotin, or avidin). Such probes are encompassed by the presentinvention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having peroxygenase activity. Genomic or other DNAfrom such other strains may be separated by agarose or polyacrylamidegel electrophoresis, or other separation techniques. DNA from thelibraries or the separated DNA may be transferred to and immobilized onnitrocellulose or other suitable carrier material. In order to identifya clone or DNA that hybridizes with SEQ ID NO: 1, or a subsequencethereof, the carrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQID NO: 1; (iii) the cDNA sequence thereof; (iv) the full-lengthcomplement thereof; or (v) a subsequence thereof; under very low to veryhigh stringency conditions. Molecules to which the nucleic acid probehybridizes under these conditions can be detected using, for example,X-ray film or any other detection means known in the art.

In one aspect, the nucleic acid probe is nucleotides 396 to 415 of SEQID NO: 1. In another aspect, the nucleic acid probe is a polynucleotidethat encodes the polypeptide of SEQ ID NO: 2; the mature polypeptidethereof; or a fragment thereof. In another aspect, the nucleic acidprobe is SEQ ID NO: 1, or the cDNA sequence thereof.

In another embodiment, the present invention relates to an isolatedpolypeptide having peroxygenase activity encoded by a polynucleotidehaving a sequence identity to the mature polypeptide coding sequence ofSEQ ID NO: 1 or the cDNA sequence thereof of at least 60%, e.g., atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO: 2 comprising a substitution, deletion,and/or insertion at one or more (e.g., several) positions. In anembodiment, the number of amino acid substitutions, deletions and/orinsertions introduced into the mature polypeptide of SEQ ID NO: 2 is notmore than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9. The amino acid changesmay be of a minor nature, that is conservative amino acid substitutionsor insertions that do not significantly affect the folding and/oractivity of the protein; small deletions, typically of 1-30 amino acids;small amino- or carboxyl-terminal extensions, such as an amino-terminalmethionine residue; a small linker peptide of up to 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for peroxygenase activity to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction, orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver etal., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acidscan also be inferred from an alignment with a related polypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

Sources of Polypeptides Having Peroxygenase Activity

A polypeptide having peroxygenase activity of the present invention maybe obtained from microorganisms of any genus. For purposes of thepresent invention, the term “obtained from” as used herein in connectionwith a given source shall mean that the polypeptide encoded by apolynucleotide is produced by the source or by a strain in which thepolynucleotide from the source has been inserted. In one aspect, thepolypeptide obtained from a given source is secreted extracellularly.

The polypeptide may be a fungal polypeptide, such as a polypeptide froma fungus in the family Chaetomiaceae. For example, the polypeptide maybe a Myceliophthora polypeptide, such as a Myceliophthora fergusii(synonym Corynascus thermophiles), Myceliophthora hinnulea,Myceliophthora lutea, Myceliophthora vellerea, or Myceliophthorathermophila polypeptide.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding thepolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

A polynucleotide may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide priorto its insertion into a vector may be desirable or necessary dependingon the expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present invention. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter from an Aspergillus neutral alpha-amylasegene in which the untranslated leader has been replaced by anuntranslated leader from an Aspergillus triose phosphate isomerase gene;non-limiting examples include modified promoters from an Aspergillusniger neutral alpha-amylase gene in which the untranslated leader hasbeen replaced by an untranslated leader from an Aspergillus nidulans orAspergillus oryzae triose phosphate isomerase gene); and mutant,truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae riose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory systems are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysystems in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used. Other examples of regulatorysequences are those that allow for gene amplification. In eukaryoticsystems, these regulatory sequences include the dihydrofolate reductasegene that is amplified in the presence of methotrexate, and themetallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the polypeptide would be operablylinked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more convenient restrictionsites to allow for insertion or substitution of the polynucleotideencoding the polypeptide at such sites. Alternatively, thepolynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMR1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the production of a polypeptide of thepresent invention. A construct or vector comprising a polynucleotide isintroduced into a host cell so that the construct or vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.In a preferred aspect, the cell is a Myceliophthora cell. In a morepreferred aspect, the cell is a Myceliophthora fergusii cell.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods known in the art. Forexample, the cell may be cultivated by shake flask cultivation, orsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe polypeptide to be expressed and/or isolated. The cultivation takesplace in a suitable nutrient medium comprising carbon and nitrogensources and inorganic salts, using procedures known in the art. Suitablemedia are available from commercial suppliers or may be preparedaccording to published compositions (e.g., in catalogues of the AmericanType Culture Collection). If the polypeptide is secreted into thenutrient medium, the polypeptide can be recovered directly from themedium. If the polypeptide is not secreted, it can be recovered fromcell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods include, but arenot limited to, use of specific antibodies, formation of an enzymeproduct, or disappearance of an enzyme substrate. For example, an enzymeassay may be used to determine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing the polypeptide is used asa source of the polypeptide.

Plants

The present invention also relates to isolated plants, e.g., atransgenic plant, plant part, or plant cell, comprising a polynucleotideof the present invention so as to express and produce a polypeptide ordomain in recoverable quantities. The polypeptide or domain may berecovered from the plant or plant part. Alternatively, the plant orplant part containing the polypeptide or domain may be used as such forimproving the quality of a food or feed, e.g., improving nutritionalvalue, palatability, and rheological properties, or to destroy anantinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing the polypeptide or domainmay be constructed in accordance with methods known in the art. Inshort, the plant or plant cell is constructed by incorporating one ormore expression constructs encoding the polypeptide or domain into theplant host genome or chloroplast genome and propagating the resultingmodified plant or plant cell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide or domain operablylinked with appropriate regulatory sequences required for expression ofthe polynucleotide in the plant or plant part of choice. Furthermore,the expression construct may comprise a selectable marker useful foridentifying plant cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide or domainis desired to be expressed. For instance, the expression of the geneencoding a polypeptide or domain may be constitutive or inducible, ormay be developmental, stage or tissue specific, and the gene product maybe targeted to a specific tissue or plant part such as seeds or leaves.Regulatory sequences are, for example, described by Tague et al., 1988,Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, or therice actin 1 promoter may be used (Franck et al., 1980, Cell 121:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay be induced by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide or domain in the plant. For instance, thepromoter enhancer element may be an intron that is placed between thepromoter and the polynucleotide encoding a polypeptide or domain. Forinstance, Xu et al., 1993, supra, disclose the use of the first intronof the rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Agrobacterium tumefaciens-mediated gene transfer is a method forgenerating transgenic dicots (for a review, see Hooykas andSchilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for transformingmonocots, although other transformation methods may be used for theseplants. A method for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5:158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternativemethod for transformation of monocots is based on protoplasttransformation as described by Omirulleh et al., 1993, Plant Mol. Biol.21: 415-428. Additional transformation methods include those describedin U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which are hereinincorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct of the present invention, transgenic plants may be made bycrossing a plant having the construct to a second plant lacking theconstruct. For example, a construct encoding a polypeptide or domain canbe introduced into a particular plant variety by crossing, without theneed for ever directly transforming a plant of that given variety.Therefore, the present invention encompasses not only a plant directlyregenerated from cells which have been transformed in accordance withthe present invention, but also the progeny of such plants. As usedherein, progeny may refer to the offspring of any generation of a parentplant prepared in accordance with the present invention. Such progenymay include a DNA construct prepared in accordance with the presentinvention. Crossing results in the introduction of a transgene into aplant line by cross pollinating a starting line with a donor plant line.Non-limiting examples of such steps are described in U.S. Pat. No.7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a polypeptideor domain of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encodingthe polypeptide or domain under conditions conducive for production ofthe polypeptide or domain; and (b) recovering the polypeptide or domain.

Removal or Reduction of Peroxygenase Activity

The present invention also relates to methods of producing a mutant of aparent cell, which comprises disrupting or deleting a polynucleotide, ora portion thereof, encoding a polypeptide of the present invention,which results in the mutant cell producing less of the polypeptide thanthe parent cell when cultivated under the same conditions.

The mutant cell may be constructed by reducing or eliminating expressionof the polynucleotide using methods well known in the art, for example,insertions, disruptions, replacements, or deletions. In a preferredaspect, the polynucleotide is inactivated. The polynucleotide to bemodified or inactivated may be, for example, the coding region or a partthereof essential for activity, or a regulatory element required forexpression of the coding region. An example of such a regulatory orcontrol sequence may be a promoter sequence or a functional partthereof, i.e., a part that is sufficient for affecting expression of thepolynucleotide. Other control sequences for possible modificationinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, signal peptide sequence, transcription terminator,and transcriptional activator.

Modification or inactivation of the polynucleotide may be performed bysubjecting the parent cell to mutagenesis and selecting for mutant cellsin which expression of the polynucleotide has been reduced oreliminated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and screeningand/or selecting for mutant cells exhibiting reduced or no expression ofthe gene.

Modification or inactivation of the polynucleotide may be accomplishedby insertion, substitution, or deletion of one or more nucleotides inthe gene or a regulatory element required for transcription ortranslation thereof. For example, nucleotides may be inserted or removedso as to result in the introduction of a stop codon, the removal of thestart codon, or a change in the open reading frame. Such modification orinactivation may be accomplished by site-directed mutagenesis or PCRgenerated mutagenesis in accordance with methods known in the art.Although, in principle, the modification may be performed in vivo, i.e.,directly on the cell expressing the polynucleotide to be modified, it ispreferred that the modification be performed in vitro as exemplifiedbelow.

An example of a convenient way to eliminate or reduce expression of apolynucleotide is based on techniques of gene replacement, genedeletion, or gene disruption. For example, in the gene disruptionmethod, a nucleic acid sequence corresponding to the endogenouspolynucleotide is mutagenized in vitro to produce a defective nucleicacid sequence that is then transformed into the parent cell to produce adefective gene. By homologous recombination, the defective nucleic acidsequence replaces the endogenous polynucleotide. It may be desirablethat the defective polynucleotide also encodes a marker that may be usedfor selection of transformants in which the polynucleotide has beenmodified or destroyed. In an aspect, the polynucleotide is disruptedwith a selectable marker such as those described herein.

The present invention also relates to methods of inhibiting theexpression of a polypeptide having peroxygenase activity in a cell,comprising administering to the cell or expressing in the cell adouble-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises asubsequence of a polynucleotide of the present invention. In a preferredaspect, the dsRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore duplex nucleotides in length.

The dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA(miRNA). In a preferred aspect, the dsRNA is small interfering RNA forinhibiting transcription. In another preferred aspect, the dsRNA ismicro RNA for inhibiting translation.

The present invention also relates to such double-stranded RNA (dsRNA)molecules, comprising a portion of the mature polypeptide codingsequence of SEQ ID NO: 1 for inhibiting expression of the polypeptide ina cell. While the present invention is not limited by any particularmechanism of action, the dsRNA can enter a cell and cause thedegradation of a single-stranded RNA (ssRNA) of similar or identicalsequences, including endogenous mRNAs. When a cell is exposed to dsRNA,mRNA from the homologous gene is selectively degraded by a processcalled RNA interference (RNAi).

The dsRNAs of the present invention can be used in gene-silencing. Inone aspect, the invention provides methods to selectively degrade RNAusing a dsRNAi of the present invention. The process may be practiced invitro, ex vivo or in vivo. In one aspect, the dsRNA molecules can beused to generate a loss-of-function mutation in a cell, an organ or ananimal. Methods for making and using dsRNA molecules to selectivelydegrade RNA are well known in the art; see, for example, U.S. Pat. Nos.6,489,127; 6,506,559; 6,511,824; and 6,515,109.

The present invention further relates to a mutant cell of a parent cellthat comprises a disruption or deletion of a polynucleotide encoding thepolypeptide or a control sequence thereof or a silenced gene encodingthe polypeptide, which results in the mutant cell producing less of thepolypeptide or no polypeptide compared to the parent cell.

The polypeptide-deficient mutant cells are particularly useful as hostcells for expression of native and heterologous polypeptides. Therefore,the present invention further relates to methods of producing a nativeor heterologous polypeptide, comprising (a) cultivating the mutant cellunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide. The term “heterologous polypeptides” meanspolypeptides that are not native to the host cell, e.g., a variant of anative protein. The host cell may comprise more than one copy of apolynucleotide encoding the native or heterologous polypeptide.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallyperoxygenase-free product is of particular interest in the production ofeukaryotic polypeptides, in particular fungal proteins such as enzymes.The peroxygenase-deficient cells may also be used to expressheterologous proteins of pharmaceutical interest such as hormones,growth factors, receptors, and the like. The term “eukaryoticpolypeptides” includes not only native polypeptides, but also thosepolypeptides, e.g., enzymes, which have been modified by amino acidsubstitutions, deletions or additions, or other such modifications toenhance activity, thermostability, pH tolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from peroxygenase activity that is produced by a methodof the present invention.

Compositions

The peroxygenase polypeptides of the invention may be added to and thusbecome a component of a detergent composition.

The detergent composition of the present invention may be formulated,for example, as a hand or machine laundry detergent compositionincluding a laundry additive composition suitable for pre-treatment ofstained fabrics and a rinse added fabric softener composition, or beformulated as a detergent composition for use in general household hardsurface cleaning operations, or be formulated for hand or machinedishwashing operations.

In a specific aspect, the present invention provides a detergentadditive comprising a polypeptide of the invention as described herein.

The detergent composition may comprise one or more surfactants, whichmay be anionic and/or cationic and/or non-ionic and/or semi-polar and/orzwitterionic, or a mixture thereof. In a particular embodiment, thedetergent composition includes a mixture of one or more nonionicsurfactants and one or more anionic surfactants. The surfactant(s) istypically present at a level of from about 0.1% to 60% by weight, suchas about 1% to about 40%, or about 3% to about 20%, or about 3% to about10%. The surfactant(s) is chosen based on the desired cleaningapplication, and includes any conventional surfactant(s) known in theart.

When included therein the detergent will usually contain from about 1%to about 40% by weight, such as from about 5% to about 30%, includingfrom about 5% to about 15%, or from about 20% to about 25% of an anionicsurfactant. Non-limiting examples of anionic surfactants includesulfates and sulfonates, in particular, linear alkylbenzenesulfonates(LAS), isomers of LAS, branched alkylbenzenesulfonates (BABS),phenylalkanesulfonates, alpha-olefinsulfonates (AOS), olefin sulfonates,alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonatesand disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate(SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS),alcohol ethersulfates (AES or AEOS or FES, also known as alcoholethoxysulfates or fatty alcohol ether sulfates), secondaryalkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates,sulfonated fatty acid glycerol esters, alpha-sulfo fatty acid methylesters (alpha-SFMe or SES) including methyl ester sulfonate (MES),alkyl- or alkenylsuccinic acid, dodecenyl/tetradecenyl succinic acid(DTSA), fatty acid derivatives of amino acids, diesters and monoestersof sulfo-succinic acid or soap, and combinations thereof.

When included therein the detergent will usually contain from about 0.2%to about 40% by weight of a non-ionic surfactant, for example from about0.5% to about 30%, in particular from about 1% to about 20%, from about3% to about 10%, such as from about 3% to about 5%, or from about 8% toabout 12%. Non-limiting examples of non-ionic surfactants includealcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylatedfatty alcohols (PFA), alkoxylated fatty acid alkyl esters, such asethoxylated and/or propoxylated fatty acid alkyl esters, alkylphenolethoxylates (APE), nonylphenol ethoxylates (NPE), alkylpolyglycosides(APG), alkoxylated amines, fatty acid monoethanolamides (FAM), fattyacid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides(EFAM), propoxylated fatty acid monoethanolamide (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine(glucamides, GA, or fatty acid glucamide, FAGA), as well as productsavailable under the trade names SPAN and TWEEN, and combinationsthereof.

The detergent composition may contain about 0-65% by weight of adetergent builder or co-builder, or a mixture thereof. In a dish washdetergent, the level of builder is typically 40-65%, particularly50-65%. The builder and/or co-builder may particularly be a chelatingagent that forms water-soluble complexes with Ca and Mg. Any builderand/or co-builder known in the art for use in laundry detergents may beutilized. Non-limiting examples of builders include zeolites,diphosphates (pyrophosphates), triphosphates such as sodium triphosphate(STP or STPP), carbonates such as sodium carbonate, soluble silicatessuch as sodium metasilicate, layered silicates (e.g., SKS-6 fromHoechst), ethanolamines such as 2-aminoethan-1-ol (MEA), iminodiethanol(DEA) and 2,2′,2″-nitrilotriethanol (TEA), and carboxymethylinulin(CMI), and combinations thereof.

The detergent composition may contain 0-50% by weight of a bleachingsystem. Any bleaching system known in the art for use in laundrydetergents may be utilized. Suitable bleaching system components includebleaching catalysts, photobleaches, bleach activators, sources ofhydrogen peroxide such as sodium percarbonate and sodium perborates,preformed peracids and mixtures thereof. Suitable preformed peracidsinclude, but are not limited to, peroxycarboxylic acids and salts,percarbonic acids and salts, perimidic acids and salts,peroxymonosulfuric acids and salts, for example, Oxone (R), and mixturesthereof. Non-limiting examples of bleaching systems includeperoxide-based bleaching systems, which may comprise, for example, aninorganic salt, including alkali metal salts such as sodium salts ofperborate (usually mono- or tetra-hydrate), percarbonate, persulfate,perphosphate, persilicate salts, in combination with a peracid-formingbleach activator. By Bleach activator is meant herein a compound whichreacts with peroxygen bleach like hydrogen peroxide to form a Peracid.The peracid thus formed constitutes the activated bleach. Suitablebleach activators to be used herein include those belonging to the classof esters amides, imides or anhydrides, Suitable examples are tetracetylathylene diamine (TAED), sodium 3,5,5 trimethyl hexanoyloxybenzenesulphonat, diperoxy dodecanoic acid, 4-(dodecanoyloxy)benzenesulfonate(LOBS), 4-(decanoyloxy)benzenesulfonate, 4-(decanoyloxy)benzoate (DOBS),4-(3,5,5-trimethylhexanoyloxy)benzenesulfonate (ISONOBS),tetraacetylethylenediamine (TAED) and 4-(nonanoyloxy)benzenesulfonate(NOBS), and/or those disclosed in WO98/17767. A particular family ofbleach activators of interest was disclosed in EP624154 and particularlypreferred in that family is acetyl triethyl citrate (ATC). ATC or ashort chain triglyceride like Triacin has the advantage that it isenvironmental friendly as it eventually degrades into citric acid andalcohol. Furthermore acethyl triethyl citrate and triacetin has a goodhydrolytical stability in the product upon storage and it is anefficient bleach activator. Finally ATC provides a good buildingcapacity to the laundry additive. Alternatively, the bleaching systemmay comprise peroxyacids of, for example, the amide, imide, or sulfonetype. The bleaching system may also comprise peracids such as6-(phthaloylamino)percapronic acid (PAP). The bleaching system may alsoinclude a bleach catalyst.

Other ingredients of the detergent composition, which are all well-knownin art, include hydrotropes, fabric hueing agents, anti-foaming agents,soil release polymers, anti-redeposition agents etc.

The detergent additive as well as the detergent composition may compriseone or more additional enzymes such as a protease, lipase, cutinase,amylase, carbohydrase, cellulase, pectinase, mannanase, arabinase,galactanase, xylanase, oxidase, e.g., a laccase, and/or peroxidase.

The polypeptide of the present invention may be added to a detergentcomposition in an amount corresponding to 0.001-100 mg of protein, suchas 0.01-100 mg of protein, preferably 0.005-50 mg of protein, morepreferably 0.01-25 mg of protein, even more preferably 0.05-10 mg ofprotein, most preferably 0.05-5 mg of protein, and even most preferably0.01-1 mg of protein per liter of wash liquor.

The polypeptide having peroxygenase activity (the peroxygenase), andoptionally also a source of hydrogen peroxide, may be formulated as aliquid (e.g. aqueous), a solid, a gel, a paste or a dry productformulation. The dry product formulation may subsequently be re-hydratedto form an active liquid or semi-liquid formulation usable in themethods of the invention.

When the peroxygenase and the source of hydrogen peroxide are formulatedas a dry formulation, the components may be mixed, arranged in discretelayers or packaged separately.

When other than dry form formulations are used, and even in that case,it is preferred to use a two-part formulation system having theperoxygenase separate from the source of hydrogen peroxide.

The composition of the invention may further comprise auxiliary agentssuch as wetting agents, thickening agents, buffer(s) for pH control,stabilisers, perfume, colourants, fillers and the like.

Useful wetting agents are surfactants, i.e. non-ionic, anionic,amphoteric or zwitterionic surfactants. Surfactants are furtherdescribed above.

Methods and Uses

The peroxygenase polypeptides of the invention may be used for sitespecific hydroxylation in position 2 or position 3 of an aliphatichydrocarbon. The aliphatic hydrocarbon must include a chain of at least3 carbons, and either (one or more) end of the aliphatic hydrocarbon maybe used as the starting point to determine which carbon is in position 2or 3. The aliphatic hydrocarbon must have at least one hydrogen attachedto the carbon (which is hydroxylated) in position 2 or 3. In a preferredembodiment, the carbon in position 2 or 3, which is hydroxylated withthe peroxygenase, is unsubstituted (before the hydroxylation is carriedout).

Accordingly, in a first aspect, the present invention provides a methodfor hydroxylation in position 2 or 3 of either end (one or more ends) ofa substituted or unsubstituted, linear or branched, aliphatichydrocarbon having at least 3 carbons and having a hydrogen attached tothe carbon in position 2 or 3, comprising contacting the aliphatichydrocarbon with hydrogen peroxide and a polypeptide having peroxygenaseactivity of the invention.

The method of the invention may be used for a variety of purposes, likebulk chemical synthesis (biocatalysis), increasing aqueous solubility ofaliphatic hydrocarbons, bioremediation, and modification of thecharacteristics of food products.

The method of the invention may also be used for a number of industrialprocesses in which said hydroxylation reactions are beneficial. Anexample of such use is in the manufacture of pulp and paper productswhere alkanes and other relevant aliphatic hydrocarbons that are presentin the wood (resin) can result in depositioning problems in the pulp andpaper manufacturing process. These hydrophobic compounds are theprecursors of the so-called pitch deposits within the pulp and papermanufacturing processes. Pitch deposition results in low quality pulp,and can cause the shutdown of pulp mill operations. Specific issuesrelated to pulps with high extractives content include runnabilityproblems, spots and holes in the paper, and sheet breaks. Treatment withperoxygenase can increase the solubility of said compounds and therebymitigate problems.

Yet another use of the method of the invention is in i.e. oil or coalrefineries where the peroxygenase catalyzed hydroxylation can be used tomodify the solubility, viscosity and/or combustion characteristics ofhydrocarbons. Specifically the treatment can lead to changes in thesmoke point, the kindling point, the fire point and the boiling point ofthe hydrocarbons subjected to the treatment.

In the synthesis of bulk chemicals, agro chemicals (incl. pesticides),specialty chemicals and pharmaceuticals the method of the invention mayobviously be relevant in terms of selectively introducing hydroxy groupsin the substrates thereby affecting the solubility of the modifiedcompound. Furthermore, the selective hydroxylation provides a site forfurther modification by methods known in the art of organic chemicalsynthesis and chemo-enzymatic synthesis.

Natural gas is extensively processed to remove higher alkanes.Hydroxylation of such higher alkanes may be used to improve watersolubility, and thus facilitate removal of the higher alkanes by washingthe natural gas stream. Removal may be performed at the well or duringrefining.

Hydroxylation of oil waste will significantly improve biodegradabilityand will be applicable both in connection with waste water treatmentfrom refineries and bioremediation of contaminated ground or water.

In a second aspect, the present invention provides a method forhydroxylation in position 2 or 3 of the terminal end of an acyl group ofa lipid, comprising contacting the lipid with hydrogen peroxide and apolypeptide having peroxygenase activity of the invention.

Hydroxylation of the acyl group of a lipid generally improves theaqueous solubility of the lipid. Accordingly, the method of theinvention may be used to remove or reduce oil or lipid containingstains, like chocolate, from laundry, by contacting the laundry with aperoxygenase and a source of hydrogen peroxide, and optionally asurfactant.

In another aspect, the methods of the invention may be used to reduceunpleasant odors from laundry by contacting the laundry with aperoxygenase and a source of hydrogen peroxide, and optionally asurfactant. The method of the invention results in reduction of theamount of butanoic acid (butyric acid) in the laundry. Butanoic acid isformed during washing of laundry when certain animal fats and plant oilsare hydrolyzed, e.g. by detergent lipase, to yield free fatty acids,including butanoic acid. Butanoic acid has an extremely unpleasant odor.The peroxygenase hydroxylates the butanoic acid to 2-hydroxybutyric acid(alpha-hydroxybutyric acid) or 3-hydroxybutyric acid(beta-hydroxybutyric acid).

The present invention also provides a method for site specificintroduction of a hydroxy and/or an oxo (keto) group at the second orthird carbon of at least two ends of an aliphatic hydrocarbon, using aperoxygenase polypeptide of the invention, and hydrogen peroxide.

The aliphatic hydrocarbon must include a chain of at least five carbons.The second and third carbons are determined by counting the carbon atomsfrom any end of the aliphatic hydrocarbon.

The aliphatic hydrocarbon must have at least one hydrogen attached to acarbon which is hydroxylated by attachment of a hydroxy group; and atleast two hydrogens attached to a carbon when an oxo group isintroduced. In a preferred embodiment, the second or third carbon isunsubstituted before being contacted with the peroxygenase.

According to the method of the invention, the hydroxy and/or oxo groupsare introduced independently of each other at the (at least) two ends ofthe aliphatic hydrocarbon. Thus, a hydroxy group can be introduced atone end, at the same time as an oxo group is introduced at another (theother) end—and vice versa. Two hydroxy groups, or two oxo groups, or onehydroxy group and one oxo group, cannot be introduced at the same end ofthe aliphatic hydrocarbon.

In the context of the present invention, “oxidation” means introductionof a hydroxy and/or an oxo group.

Accordingly, in a first aspect, the present invention provides a methodfor introducing a hydroxy and/or an oxo (keto) group at the second orthird carbon of (at least) two ends of a substituted or unsubstituted,linear or branched, aliphatic hydrocarbon having at least five carbonsand having at least one hydrogen attached to said second or thirdcarbon, comprising contacting the aliphatic hydrocarbon with hydrogenperoxide and a polypeptide having peroxygenase activity of theinvention.

In a preferred embodiment, the aliphatic hydrocarbon is oxidized to(converted to) a diol, by introduction of two hydroxy groups. Morepreferably, the two hydroxy groups are located at each end of a linearaliphatic hydrocarbon.

The method of the invention may be used for a variety of purposes, likebulk chemical synthesis (biocatalysis), increasing aqueous solubility ofaliphatic hydrocarbons, bioremediation, and modification of thecharacteristics of food products.

The method of the invention may also be used for a number of industrialprocesses in which said oxidation reactions are beneficial. An exampleof such use is in the manufacture of pulp and paper products wherealkanes and other relevant aliphatic hydrocarbons that are present inthe wood (resin) can result in depositioning problems in the pulp andpaper manufacturing process. These hydrophobic compounds are theprecursors of the so-called pitch deposits within the pulp and papermanufacturing processes. Pitch deposition results in low quality pulp,and can cause the shutdown of pulp mill operations. Specific issuesrelated to pulps with high extractives content include runnabilityproblems, spots and holes in the paper, and sheet breaks. Treatment withperoxygenase can increase the solubility of said compounds and therebymitigate problems.

Yet another use of the method of the invention is in, for example, oilor coal refineries where the peroxygenase catalyzed oxidation can beused to modify the solubility, viscosity and/or combustioncharacteristics of hydrocarbons. Specifically the treatment can lead tochanges in the smoke point, the kindling point, the fire point and theboiling point of the hydrocarbons subjected to the treatment.

In the synthesis of bulk chemicals, agro chemicals (incl. pesticides),specialty chemicals and pharmaceuticals the method of the invention mayobviously be relevant in terms of selectively introducing hydroxy groupsin the substrates thereby affecting the solubility of the modifiedcompound. Furthermore, the selective oxidation provides a site forfurther modification by methods known in the art of organic chemicalsynthesis and chemo-enzymatic synthesis.

Natural gas is extensively processed to remove higher alkanes. Oxidationof such higher alkanes may be used to improve water solubility, and thusfacilitate removal of the higher alkanes by washing the natural gasstream. Removal may be performed at the well or during refining.

Oxidation, according to the invention, of oil waste will significantlyimprove biodegradability and will be applicable both in connection withwaste water treatment from refineries and bioremediation of contaminatedground or water.

The methods of the invention may be carried out with an immobilizedperoxygenase polypeptide of the invention.

The methods of the invention may be carried out in an aqueous solvent(reaction medium), various alcohols, ethers, other polar or non-polarsolvents, or mixtures thereof. By studying the characteristics of thealiphatic hydrocarbon used in the methods of the invention, suitableexamples of solvents are easily recognized by one skilled in the art. Byraising or lowering the pressure at which the hydroxylation/oxidation iscarried out, the solvent (reaction medium) and the aliphatic hydrocarboncan be maintained in a liquid phase at the reaction temperature.

The methods according to the invention may be carried out at atemperature between 0 and 90 degrees Celsius, preferably between 5 and80 degrees Celsius, more preferably between 10 and 70 degrees Celsius,even more preferably between 15 and 60 degrees Celsius, most preferablybetween 20 and 50 degrees Celsius, and in particular between 20 and 40degrees Celsius.

The methods of the invention may employ a treatment time of from 10seconds to (at least) 24 hours, preferably from 1 minute to (at least)12 hours, more preferably from 5 minutes to (at least) 6 hours, mostpreferably from 5 minutes to (at least) 3 hours, and in particular from5 minutes to (at least) 1 hour.

Diols (di-hydroxy aliphatic hydrocarbons) produced by the method of theinvention may be used for producing polyurethan. Polyurethane is apolymer composed of a chain of organic units joined by carbamate(urethane) links. Polyurethane polymers are formed through step-growthpolymerization, by reacting a monomer (with at least two isocyanatefunctional groups) with another monomer (with at least two hydroxylgroups) in the presence of a catalyst.

The present invention also provides a method for introducing an oxo(keto) group at the second or third carbon of a substituted orunsubstituted, linear or branched, aliphatic hydrocarbon having at leastfive carbons and having at least two hydrogens attached to said secondor third carbon, comprising contacting the aliphatic hydrocarbon withhydrogen peroxide and a polypeptide having peroxygenase activity of theinvention.

In yet another aspect, the present invention also provides a method forintroducing a hydroxy or an oxo group at a terminal carbon of a linearor branched aliphatic hydrocarbon having at least five carbons, which issubstituted with a carboxy group, comprising contacting the aliphatichydrocarbon with hydrogen peroxide and a polypeptide having peroxygenaseactivity of the invention.

In an embodiment, the aliphatic hydrocarbon which is substituted with acarboxy group is a fatty acid; preferably butanoic acid (butyric acid),pentanoic acid (valeric acid), hexanoic acid (caproic acid), heptanoicacid (enanthic acid), octanoic acid (caprylic acid), nonanoic acid(pelargonic acid), decanoic acid (capric acid), dodecanoic acid (lauricacid), tetradecanoic acid (myristic acid), hexadecanoic acid (palmiticacid), octadecanoic acid (stearic acid), eicosanoic acid (arachidicacid), linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoicacid, or docosahexaenoic acid.

In yet another aspect, the present invention also provides a method forchanging (oxidizing) a primary alcohol of a linear or branched aliphatichydrocarbon having at least five carbons to the corresponding acid,comprising contacting the alcohol of an aliphatic hydrocarbon withhydrogen peroxide and a polypeptide having peroxygenase activity of theinvention.

For example, pentanol may be changed (oxidized) to pentanoic acid(valeric acid), hexanol may be changed to hexanoic acid (caproic acid),heptanol may be changed to heptanoic acid (enanthic acid), octanol maybe changed to octanoic acid (caprylic acid), nonanol may be changed tononanoic acid (pelargonic acid), decanol may be changed to decanoic acid(capric acid), dodecanol may be changed to dodecanoic acid (lauricacid), tetradecanol may be changed to tetradecanoic acid (myristicacid), hexadecanol may be changed to hexadecanoic acid (palmitic acid),octadecanol may be changed to octadecanoic acid (stearic acid), andeicosanol may be changed to eicosanoic acid (arachidic acid).

The polypeptides having peroxygenase activity of the invention(peroxygenase polypeptides or peroxygenases) are used in the methods ofthe invention in an amount of 0.005-50 ppm (mg/I), or 0.01-40, 0.02-30,0.03-25, 0.04-20, 0.05-15, 0.05-10, 0.05-5, 0.05-1, 0.05-0.8, 0.05-0.6,or 0.1-0.5 ppm. The amount of enzyme refers to mg of a well-definedenzyme preparation.

In the methods of the invention, the peroxygenase may be applied aloneor together with an additional enzyme. The term “an additional enzyme”means at least one additional enzyme, e.g. one, two, three, four, five,six, seven, eight, nine, ten or even more additional enzymes.

The term “applied together with” (or “used together with”) means thatthe additional enzyme may be applied in the same, or in another step ofthe method of the invention. The other process step may be upstream ordownstream, as compared to the step in which the peroxygenase is used.

In particular embodiments the additional enzyme is an enzyme which hasprotease, lipase, xylanase, cutinase, oxidoreductase, cellulase,endoglucanase, amylase, mannanase, steryl esterase, and/or cholesterolesterase activity. Examples of oxidoreductase enzymes are enzymes withlaccase, and/or peroxidase activity.

The term “a step” of a method means at least one step, and it could beone, two, three, four, five or even more method steps. In other wordsthe peroxygenases of the invention may be applied in at least one methodstep, and the additional enzyme(s) may also be applied in at least onemethod step, which may be the same or a different method step ascompared to the step where the peroxygenase is used.

The term “enzyme preparation” means a product containing at least oneperoxygenase. The enzyme preparation may also comprise enzymes havingother enzyme activities. In addition to the enzymatic activity, such apreparation preferably contains at least one adjuvant. Examples ofadjuvants are buffers, polymers, surfactants and stabilizing agents.

Hydrogen Peroxide

The hydrogen peroxide (or source of hydrogen peroxide) required by theperoxygenase may be provided as an aqueous solution of hydrogen peroxideor a hydrogen peroxide precursor for in situ production of hydrogenperoxide. Any solid entity which liberates upon dissolution a peroxidewhich is useable by peroxygenase can serve as a source of hydrogenperoxide. Compounds which yield hydrogen peroxide upon dissolution inwater or an appropriate aqueous based medium include but are not limitedto metal peroxides, percarbonates, persulphates, perphosphates,peroxyacids, alkyperoxides, acylperoxides, peroxyesters, urea peroxide,perborates and peroxycarboxylic acids or salts thereof.

Another source of hydrogen peroxide is a hydrogen peroxide generatingenzyme system, such as an oxidase together with a substrate for theoxidase. Examples of combinations of oxidase and substrate comprise, butare not limited to, amino acid oxidase (see e.g. U.S. Pat. No.6,248,575) and a suitable amino acid, glucose oxidase (see e.g. WO95/29996) and glucose, lactate oxidase and lactate, galactose oxidase(see e.g. WO 00/50606) and galactose, and aldose oxidase (see e.g. WO99/31990) and a suitable aldose.

By studying EC 1.1.3._, EC 1.2.3._, EC 1.4.3._, and EC 1.5.3._(—) orsimilar classes (under the International Union of Biochemistry), otherexamples of such combinations of oxidases and substrates are easilyrecognized by one skilled in the art.

Hydrogen peroxide or a source of hydrogen peroxide may be added at thebeginning of or during the method of the invention, e.g. as one or moreseparate additions of hydrogen peroxide; or continuously as fed-batchaddition. Typical amounts of hydrogen peroxide correspond to levels offrom 0.001 mM to 25 mM, preferably to levels of from 0.005 mM to 5 mM,and particularly to levels of from 0.01 to 1 mM hydrogen peroxide.Hydrogen peroxide may also be used in an amount corresponding to levelsof from 0.1 mM to 25 mM, preferably to levels of from 0.5 mM to 15 mM,more preferably to levels of from 1 mM to 10 mM, and most preferably tolevels of from 2 mM to 8 mM hydrogen peroxide.

Aliphatic Hydrocarbons

The hydrocarbons, which are hydroxylated in the method of the invention,are aliphatic hydrocarbons having a chain of at least 3 carbons, andhaving a hydrogen attached to the carbon in position 2 or 3. Preferably,the aliphatic hydrocarbon is an alkane or an alkene; more preferably,the aliphatic hydrocarbon is an alkane, such as propane, butane,pentane, hexane, heptane, octane, nonane or decane, or isomers thereof.

The aliphatic hydrocarbons are linear or branched, but not cyclic, assite specific hydroxylation is not possible with cyclic hydrocarbons.Branched hydrocarbons correspond to isomers of linear hydrocarbons.

The aliphatic hydrocarbons are substituted or unsubstituted. Preferably,the aliphatic hydrocarbons are unsubstituted, such as non-activatedhydrocarbons.

When the aliphatic hydrocarbons are substituted (functional groupsattached), the preferred substituents are halogen, hydroxyl, carboxyl,amino, nitro, cyano, thiol, sulphonyl, formyl, acetyl, methoxy, ethoxy,phenyl, benzyl, xylyl, carbamoyl and sulfamoyl; more preferredsubstituents are chloro, hydroxyl, carboxyl and sulphonyl; and mostpreferred substituents are chloro and carboxyl.

The aliphatic hydrocarbons may be substituted by up to 10 substituents,up to 8 substituents, up to 6 substituents, up to 4 substituents, up to2 substituents, or by up to one substituent.

In a preferred embodiment, the aliphatic hydrocarbon is a fatty acid(the substituent is a carboxyl group). Examples of fatty acids include,but are not limited to, butanoic acid (butyric acid), pentanoic acid(valeric acid), hexanoic acid (caproic acid), heptanoic acid (enanthicacid), octanoic acid (caprylic acid), nonanoic acid (pelargonic acid),decanoic acid (capric acid), dodecanoic acid (lauric acid),tetradecanoic acid (myristic acid), hexadecanoic acid (palmitic acid),octadecanoic acid (stearic acid), eicosanoic acid (arachidic acid),linoleic acid, linolenic acid, arachidonic acid, eicosapentaenoic acid,and docosahexaenoic acid.

In a second aspect, the aliphatic hydrocarbon is an acyl group of alipid, such as a monoglyceride, diglyceride, triglyceride, phospholipidor sphingolipid; and the hydroxylation takes place in position 2 orposition 3 of the terminal end of the acyl group. The acyl group musthave at least one hydrogen attached to the carbon in position 2 or 3 ofthe terminal end. The acyl group may be saturated or unsaturated, andoptionally functional groups (substituents) may be attached. Examples ofacyl groups include, but are not limited to, the acyl forms of butanoicacid (butyric acid), pentanoic acid (valeric acid), hexanoic acid(caproic acid), heptanoic acid (enanthic acid), octanoic acid (caprylicacid), nonanoic acid (pelargonic acid), decanoic acid (capric acid),dodecanoic acid (lauric acid), tetradecanoic acid (myristic acid),hexadecanoic acid (palmitic acid), octadecanoic acid (stearic acid),eicosanoic acid (arachidic acid), linoleic acid, linolenic acid,arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid.

Signal Peptide

The present invention also relates to an isolated polynucleotideencoding a signal peptide comprising or consisting of amino acids −17 to−1 of SEQ ID NO: 2. The polynucleotides may further comprise a geneencoding a protein, which is operably linked to the signal peptide. Theprotein is preferably foreign to the signal peptide. In one aspect, thepolynucleotide encoding the signal peptide is nucleotides 1 to 51 of SEQID NO: 1.

The present invention also relates to nucleic acid constructs,expression vectors and recombinant host cells comprising suchpolynucleotides.

The present invention also relates to methods of producing a protein,comprising (a) cultivating a recombinant host cell comprising suchpolynucleotide; and (b) recovering the protein.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andpolypeptides. The term “protein” also encompasses two or morepolypeptides combined to form the encoded product. The proteins alsoinclude hybrid polypeptides and fused polypeptides.

Preferably, the protein is a hormone, enzyme, receptor or portionthereof, antibody or portion thereof, or reporter. For example, theprotein may be a hydrolase, isomerase, ligase, lyase, oxidoreductase, ortransferase, e.g., an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease,endoglucanase, esterase, alpha-galactosidase, beta-galactosidase,glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase,lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase, xylanase, or beta-xylosidase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Strains

Aspergillus oryzae MT3568 is an amdS (acetamidase) disrupted genederivative of Aspergillus oryzae JaL355 (see WO 2002/40694), in whichpyrG auxotrophy was restored by disrupting the A. oryzae acetamidase(amdS) gene with the pyrG gene. Protoplasts of Aspergillus oryzae MT3568were prepared according to WO 95/02043 (“Transformation of Aspergillusoryzae or Aspergillus niger”), but using Glucanex (which is identical toLysing Enzyme, Sigma L1412) instead of Novozym® 234.

Media and Solutions

DAP-4C-1 medium

11 g MgSO₄, 7H₂O;

1.0 g KH₂PO₄;

2.0 g Citric acid (C₆H₈O₇, H₂O);

20 g Dextrose;

10 g Maltose;

5.2 g K₃PO₄, H₂O;

0.5 g Yeast Extract; and

0.5 ml KU6 Trace metal solution (AMG, MSA-SUB-FS-0042).

Add 500 ml Milli-Q-water and mix until completely dissolved.

1 ml Dowfax 63N10 (linear EO/PO block copolymers, defoam/antifoam agent)is added.

Adjust volume with Milli-Q-water up to 1000 ml.

Add CaCO₃ tablets á 0.5 g (add 1 tablet per 200 ml).

Before inoculation, each shake flask á 150 ml is added 3.5 ml of 50%di-ammoniumhydrogenphosphat ((NH₄)₂HPO₄), and 5.0 ml of 20% lactic acid.

KU6 Trace Metal Solution (AMG, MSA-SUB-FS-0042)

6.8 g ZnCl₂;

2.5 g CuSO₄, 5H₂O;

0.13 g Nickel Chloride anhydrous;

13.9 g FeSO₄, 7H₂O;

8.45 g MnSO₄, H₂O;

3.0 g Citric acid (C₆H₈O₇, H₂O); and

Ion exchanged water up to 1000 ml.

TABLE 1 KU6 trace metal solution. Raw material Chem. formula Supplier7-cif. no. Amount Zinc Chloride ZnCl₂ Merck 108816 102-4965 6.8 g CopperSulfate CuSO₄, 5H₂O Merck 102790 109-0771 2.5 g Nickel NiCl₂ Merck806722 101-6652 0.13 g Chloride anhydrous Iron Sulfate FeSO₄, 7H₂O Merck103965 13.9 g Manganese MnSO₄, H₂O Merck 105941 8.45 g Sulfate Citricacid C₆H₈O₇, H₂O Merck 100244 3.0 g Ion exchanged 1000 ml water up to

LB plates were composed of 10 g of Bacto-Tryptone, 5 g of yeast extract,10 g of sodium chloride, 15 g of Bacto-agar, and deionized water to 1liter.

LB medium was composed of 10 g of Bacto-Tryptone, 5 g of yeast extract,and 10 g of sodium chloride, and deionized water to 1 liter.

COVE sucrose plates were composed of 342 g of sucrose, 20 g of agarpowder, 20 ml of COVE salt solution, and deionized water to 1 liter. Themedium was sterilized by autoclaving at 15 psi for 15 minutes(Bacteriological Analytical Manual, 8th Edition, Revision A, 1998). Themedium was cooled to 60° C. and 10 mM acetamide, Triton X-100 (50 μl per500 ml) were added.

COVE salt solution was composed of 26 g of MgSO₄, 7H₂O; 26 g of KCl; 26g of KH₂PO₄; 50 ml of COVE trace metal solution, and deionized water to1 liter.

COVE trace metal solution was composed of 0.04 g of Na₂B₄O₇, 10H₂O; 0.4g of CuSO₄, 5H₂O; 1.2 g of FeSO₄, 7H₂O; 0.7 g of MnSO₄, H₂O; 0.8 g ofNa₂MoO₄, 2H₂O; 10 g of ZnSO₄, 7H₂O; and deionized water to 1 liter.

Example 1 Expression of a Mature Peroxygenase from MyceliophthoraFergusii

The polynucleotide of SEQ ID NO: 1 is a genomic nucleotide sequenceencoding a peroxygenase, isolated from Myceliophthora fergusii (synonymCorynascus thermophilus) CBS 174.70. Myceliophthora fergusii CBS 174.70(obtained from Centraalbureau voor Schimmelcultures, The Netherlands)originates from England, 1970.

The polynucleotide of SEQ ID NO: 1 was amplified by PCR. The PCR wascomposed of 1 μl of genomic DNA of Myceliophthora fergusii, 0.75 μl ofcloning primer forward (10 μM), 0.75 μl of cloning primer reverse (10μM), 3 μl of 5× HF buffer (Finnzymes Oy, Finland), 0.25 μl of 50 mMMgCl₂, 0.30 μl of 10 mM dNTP, 0.15 μl of PHUSION® DNA polymerase(Finnzymes Oy, Finland), and 8.8 μl PCR-grade water. The amplificationreaction was performed using a Thermal Cycler programmed for 2 minutesat 98° C. followed by 35 cycles each consisting of 98° C. for 10 secondsand 72° C. for 90 seconds; followed by a single extension at 72° C. for5 minutes.

Cloning primer forward (SEQ ID NO: 17):5′- ACACAACTGGGGATCCACCATGAGAGCTTCTATCCTGCCAGTCT T -3′Cloning primer reverse (SEQ ID NO: 18):5′- AGATCTCGAGAAGCTTAGAAACCGAAATGGGTGCGCCG -3′

The PCR product was isolated on 1.0% agarose gel electrophoresis usingTAE buffer where the PCR band was excised from the gel and purifiedusing a GFX® PCR DNA and Gel Band Purification Kit (GE Healthcare,HiHerød Denmark) according to manufacturer's instructions. DNAcorresponding to the Myceliophthora fergusii peroxygenase gene (SEQ IDNO: 1) was cloned into the expression vector pDAu109 (see WO2005/042735) previously linearized with Bam HI and Hind III, using anIN-FUSION™ Dry-Down PCR Cloning Kit (BD Biosciences, Palo Alto, Calif.,USA) according to the manufacturer's instructions.

A 1 μl volume of the undiluted ligation mixture was used to transform E.coli TOP10 chemically competent cells (Invitrogen, Carlsbad, Calif.,USA). Two colonies were selected on LB agar plates containing 100 μg ofampicillin per ml and cultivated overnight in 2 ml of LB mediumsupplemented with 100 μg of ampicillin per ml. Plasmid DNA was purifiedusing an Jetquick Plasmid Miniprep Spin Kit (Genomed GmbH, Løohne,Germany) according to the manufacturer's instructions.

The Myceliophthora fergusii peroxygenase gene sequence was verified bySanger sequencing before heterologous expression.

A plasmid containing the nucleotide sequence of SEQ ID NO: 1 wasselected for heterologous expression of the peroxygenase gene in anAspergillus oryzae MT3568 host cell.

A. oryzae MT3568 is an amdS (acetamidase) disrupted gene derivative ofAspergillus oryzae JaL355 (see WO 2002/40694) in which pyrG auxotrophywas restored by disrupting the A. oryzae acetamidase (amdS) gene withthe pyrG gene. Protoplasts of Aspergillus oryzae MT3568 were preparedusing Glucanex (which is identical to Lysing Enzyme, Sigma L1412).

One hundred μl of Aspergillus oryzae MT3568 protoplasts were mixed with1-2 μg of the Aspergillus expression vector containing the nucleotidesequence of SEQ ID NO: 1, and 250 μl of 60% PEG 4000 (Applichem,Darmstadt, Germany) (polyethylene glycol, molecular weight 4,000), 10 mMCaCl₂, and 10 mM Tris-HCI pH 7.5 and gently mixed. After 30 minutes ofincubation at 37° C., 4 ml of topagar (temp. 40° C.) was added, and theprotoplasts were spread onto COVE plates for selection. After incubationfor 4-7 days at 37° C. spores of four transformants were inoculated into0.5 ml of DAP-4C-01 medium in 96 deep well plates. After 4-5 dayscultivation at 30° C., the culture broths were analyzed by SDS-PAGE toidentify the transformants producing the largest amount of recombinantperoxygenase from Myceliophthora fergusii, and the culture broths werealso analyzed in assays for confirmation of activity.

Spores of the best transformant were spread on COVE plates containing0.01% TRITON® X-100 in order to isolate single colonies. The spreadingwas repeated twice.

An Aspergillus oryzae transformant constructed as described above wasfermented in 150 ml DAP-4C-01 medium in 500 ml fluted shake flasksincubated at 26-30° C. in a shaking platform incubator, rotating at 150RPM for 5 days and further used for assays as described below.

Example 2 Oxidation of 4-Nitrobenzodioxole

Peroxygenases oxidize 4-nitrobenzodioxole(1,2-(Methylenedioxy)-4-nitrobenzene) to 4-nitrocatechol and theproduced yellow color was quantified spectrophotometrically at 425 nm(ε₄₂₅=9,700 M⁻¹cm⁻¹).

A 10 mM stock solution of 4-nitrobenzodioxole (98% pure, 161500 Aldrich)was prepared in acetonitrile. A 0.1 mg/ml stock of purified peroxygenase(containing the peroxygenase of SEQ ID NO: 2) was prepared in de-ionizedwater. The final reaction mixture (0.2 mL) contained 1.0 mM4-nitrobenzodioxole, 10% acetonitrile, buffer (50 mM acetate buffer pH4.5 or 50 mM phosphate buffer pH 6.5 or 25 mM borate buffer pH 8.5),0.002 mg purified peroxygenase/ml and 0.5 mM hydrogen peroxide. Thereaction was started by addition of hydrogen peroxide. A SpectraMax Plus384 plate reader was applied (kinetics at 30° C. at 425 nm) using a 96well microtitre plate from Nunc (no. 260836). Each sample was analysedin triplicates. Blanks prepared without addition of hydrogen peroxidewere subtracted.

The increase in absorbance was recorded over 2 minutes, and the results(see Table 2) show that the peroxygenase converts 4-nitrobenzodioxole to4-nitrocatechol.

TABLE 2 Absorbance (A₄₂₅) measurements. Time (seconds) pH 4.5 pH 6.5 pH8.5 0 0.018 0.061 0.029 40 0.042 0.186 0.093 80 0.048 0.249 0.099 1200.050 0.290 0.103

Example 3 Oxidation of Veratryl Alcohol

Oxidation of 1 mM veratryl alcohol with 1 mM H₂O₂ was carried out with20% acetonitrile and 5 mM phosphate buffer at pH 6.5, using 0.01 mg/mLof purified peroxygenase (mature peroxygenase encoded by SEQ ID NO: 1)in a total reaction volume of 1 mL. The reaction was performed at roomtemperature for 25 minutes and stopped by adding 1 μL of catalase(Terminox Ultra 50L, Novozymes).

Sample was analyzed on an Agilent 1200 HPLC system equipped with a DiodeArray Detector (Agilent, Santa Clara Calif., USA) and separated on aGemini C6-Phenyl (110 Å, 2×150 mm, 3 μm) column from Phenomenex(Torrance Calif., USA) thermostated at 40° C. Two mobile phases wereused: (A) 0.1% formic acid, and (B) 0.1% formic acid in acetonitrile.

Separation was run using stepwise gradient starting with 20% B held for1 min, then increasing to 55% B within 4 min and being maintained at 55%for 1 min with a constant flow rate of 0.4 mL/min.

Veratryl alcohol and its oxidation products veratryl aldehyde andveratric acid were identified and quantified by external calibrationusing authentic standards, based on their retention times, UV absorptionspectra (230 nm, 280 nm, and 260 nm respectively).

The peroxygenase oxidised veratryl alcohol yielding a single product,veratryl aldehyde (1.0%).

Example 4 Oxidation of Dibenzothiophene

Oxidations of 1 mM dibenzothiophene with 1 mM H₂O₂ were carried out with30% acetonitrile and 10 mM acetate (pH 3-5), phosphate (pH 6-7) orborate buffer (pH 8) at specified pH values, using 0.01 mg/mL ofpurified peroxygenase (mature peroxygenase encoded by SEQ ID NO: 1) in atotal reaction volume of 1 mL. Reactions were performed at roomtemperature for 25 minutes and stopped by adding 1 μL of catalase(Terminox Ultra 50L, Novozymes).

Samples were analyzed on an Agilent 1200 HPLC system equipped with aDiode Array Detector (Agilent, Santa Clara Calif., USA) and separated ona Gemini C6-Phenyl (110 Å, 2×150 mm, 3 μm) column from Phenomenex(Torrance Calif., USA) thermostated at 40° C. Two mobile phases wereused: (A) 0.1% formic acid, and (B) 0.1% formic acid in acetonitrile.

Separations were run using stepwise gradient starting with 30% B heldfor 0.5 min, then increasing to 80% B within 14.5 min and beingmaintained at 80% for 3 min with a constant flow rate of 0.4 mL/min.

Dibenzothiophene and its oxidation product dibenzothiophene sulfone wasidentified and quantified by external calibration using authenticstandards, based on their retention times, UV absorption spectra (230 nmand 260 nm). Dibenzothiophene oxide standard was not commerciallyavailable the quantification of this compound was done usingdibenzothiophene sulfone calibration curve.

The peroxygenase oxidised dibenzothiophene yielding one product,dibenzothiophene oxide (DBT-SO).

TABLE 3 Yields of dibenzothiophene oxide at various pH. pH DBT-SO yield(%) 3 11 5 91 6 91 7 92 8 11

Example 5 Oxidation of Naphthalene

Oxidations of 1 mM naphthalene with 1 mM H₂O₂ were carried out with 20%acetonitrile and 10 mM acetate (pH 3-5), phosphate (pH 6-7) or boratebuffer (pH 8) at specified pH values, using 0.01 mg/mL of purifiedperoxygenase (mature peroxygenase encoded by SEQ ID NO: 1) in a totalreaction volume of 1 mL. Reactions were performed at room temperaturefor 25 minutes and stopped by adding 1 μL of catalase (Terminox Ultra50L, Novozymes).

Samples were analyzed on an Agilent 1200 HPLC system equipped with aDiode Array Detector (Agilent, Santa Clara Calif., USA) and separated ona Gemini C6-Phenyl (110 Å, 2×150 mm, 3 μm) column from Phenomenex(Torrance Calif., USA) thermostated at 40° C. Two mobile phases wereused: (A) 0.1% formic acid, and (B) 0.1% formic acid in acetonitrile.

Separations were run using stepwise gradient starting with 30% B heldfor 0.5 min, then increasing to 50% B within 3.5 min and then increasingto 60% B within 5 min with a constant flow rate of 0.4 mL/min.

Naphthalene and its oxidation products 1-naphthol, 2-naphthol,1,4-naphthoquinone and naphthalene-1,4-diol were identified usingauthentic standards, based on their retention times and UV absorptionspectra (210 nm or 204 nm). Quantification was done based on externalcalibration of authentic standards except for 1,4-naphthoquinone thatwas quantified using naphthalene-1,4-diol as a standard.

The peroxygenase oxidised naphthalene yielding multiple products,1-naphthol (1-NOL), 2-naphthol (2-NOL), naphthalene-1,4-diol (NPD),1,4-naphthoquinone (NPQ), and some unidentified products.

TABLE 4 Naphthalene oxidation product yields at various pH. Yield (%)Total pH 1-NOL 2-NOL NPD NPQ Unknown product 3 0 0 0 0 0 0 5 12 1 8 1 829 6 18 0 8 1 16 43 7 18 0 9 1 15 45 8 4 0 1 0 1 5

Example 6 Oxidation of Benzyl Alcohol

Oxidations of 1 mM benzyl alcohol with 2 mM H₂O₂ were carried with 30%acetonitrile and 10 mM acetate (pH 3-5), phosphate (pH 7) or boratebuffer (pH 9) at specified pH values, using 0.01 mg/mL of purifiedperoxygenase (mature peroxygenase encoded by SEQ ID NO: 1) in a totalreaction volume of 1 mL. Reactions were performed at room temperaturefor 25 minutes and stopped by adding 1 μL of catalase (Terminox Ultra50L, Novozymes).

Samples were analyzed on an Agilent 1200 HPLC system equipped with aDiode Array Detector (Agilent, Santa Clara Calif., USA) and separated ona Zorbax Stable Bond C18 (2) (80 Å, 2.1×50 mm, 1.8 μm) column fromPhenomenex (Torrance Calif., USA) thermostated at 40° C. Two mobilephases were used: (A) 0.1% formic acid, and (B) 0.1% formic acid inacetonitrile.

Separations were run using stepwise gradient starting with 5% B held for4 min, then increasing to 100% B within 6 min with a constant flow rateof 0.5 ml/min.

Benzyl alcohol and its oxidation products, benzaldehyde and benzoicacid, were identified and quantified by external calibration usingauthentic standards, based on their retention times, UV absorptionspectra (210 nm, 250 nm, and 230 nm respectively).

The peroxygenase oxidised benzyl alcohol yielding two productsbenzaldehyde and benzoic acid.

TABLE 5 Comparison of total product, benzaldehyde (B—CHO) and benzoicacid (B—COOH) yields calculated at various pH. pH B—CHO (%) B—COOH (%)Total product (%) 3 0 0 0 5 12 0 12 7 24 2 25 9 0 0 0

Example 7 Oxidation of 4-Hydroxybenzoic Acid

Oxidations of 2 mM 4-hydroxybenzoic acid with 1 mM H₂O₂ were carried outin 20% acetonitrile and 10 mM phosphate buffer at pH 6 with 2 mMascorbic acid, using 0.01 mg/mL of purified peroxygenase (matureperoxygenase encoded by SEQ ID NO: 1) in a total reaction volume of 1mL. Reactions were performed at room temperature for 15 minutes andstopped by adding 1 μL of catalase (Terminox Ultra 50L, Novozymes).

Samples were analyzed on an Agilent 1200 HPLC system equipped with aDiode Array Detector (Agilent, Santa Clara Calif., USA) and separated ona Zorbax Stable Bond C18 (2) (80 Å, 2.1×50 mm, 1.8 μm) column fromPhenomenex (Torrance Calif., USA) thermostated at 40° C. Two mobilephases were used: (A) 0.1% formic acid, and (B) 0.1% formic acid inacetonitrile.

Separations were run using stepwise gradient starting with 5% B held for4 min, then increasing to 100% B within 6 min with a constant flow rateof 0.5 ml/min.

4-hydroxybenzoic acid and its oxidation product 3,4-dihydroxybenzoicacid were identified and quantified by external calibration usingauthentic standards, based on their retention times, UV absorptionspectra at 210 nm.

The peroxygenase oxidised 4-hydroxybenzoic acid yielding a singleproduct, 3,4-dihydroxybenzoic acid (0.8%).

Example 8 Oxidation of n-Heptane

Oxidations of 2 mM n-heptane with 1 mM H₂O₂ were carried out with 20%acetone and 10 mM phosphate buffer at pH 6, using 0.01 mg/mL of purifiedperoxygenase (mature peroxygenase encoded by SEQ ID NO: 1) in a totalreaction volume of 1 mL. Reactions were performed at room temperaturefor 10 minutes and samples were then inactivated by adding extractionsolvent ethyl acetate.

Samples were analyzed on an The Agilent 7890A Gas Chromatograph equippedwith Agilent 5975C series MSD system (Agilent, Santa Clara Calif., USA)and a ZB-5HT (15×0.25 mm, 0.1 μm) column from Phenomenex (TorranceCalif., USA). Helium was used as carrier gas at a constant flow rate of2 mL/min.

For analysis, 2 μL of ethyl acetate extract was injected into GC systemat 250° C. in the split mode (50:1). Separations were run usingtemperature program starting with 45° C. held for 1 min, then increasingto 50° C. at a rate of 5° C./min and holding for 1 min, then increasingto 200° C. at a rate of 30° C./min and holding for 0.5 min.

n-Heptane oxidation products were identified and quantified by externalcalibration using authentic standards, based on their retention timesand electron impact MS at 70 eV.

The peroxygenase oxidised n-heptane yielding multiple products,2-heptanol, 3-heptanol, 2-heptanone and 3-heptanone.

TABLE 6 n-Heptane oxidation product yields. Product Yield (%) 2-Heptanol7.5 3-Heptanol 5.0 2-Heptanone 1.0 3-Heptanone 0 Total product 13.5

Example 9 Oxidation of Cyclohexane

Oxidations of 2 mM cyclohexane with 1 mM H₂O₂ were carried out with 20%acetone and 5 mM acetate (pH 3-5), phosphate (pH 6-7) or borate (pH 9)buffer at specified pH values, using 0.01 mg/mL of purified peroxygenase(mature peroxygenase encoded by SEQ ID NO: 1) in a total reaction volumeof 1 mL. Reactions were performed at room temperature for 10 minutes andsamples were then inactivated by adding extraction solvent ethylacetate.

Samples were analyzed on an The Agilent 7890A Gas Chromatograph equippedwith Agilent 5975C series MSD system (Agilent, Santa Clara Calif., USA)and a ZB-5HT (15×0.25 mm, 0.1 μm) column from Phenomenex (TorranceCalif., USA). Helium was used as carrier gas at a constant flow rate of1.2 mL/min.

For analysis 2 μL of ethyl acetate extract was injected into GC systemat 250° C. in the split mode (50:1). Separations were run usingtemperature program starting with 45° C. held for 1 min, then increasingto 90° C. at a rate of 20° C./min, and then increasing to 160° C. at arate of 35° C./min and holding for 0.5 min.

Cyclohexane oxidation products were identified and quantified byexternal calibration using authentic standards, based on their retentiontimes and electron impact MS at 70 eV.

The peroxygenase oxidised cyclohexane yielding a single product,cyclohexanone.

TABLE 7 Comparison of cyclohexanone oxidation product yields at variouspH. pH Cyclohexanone (%) 3 0.0 5 1.3 6 1.2 7 1.3 9 0.3

Example 10 Oxidation of Isobutylbenzene

Oxidation of 1 mM isobutylbenzene with 1 mM H₂O₂ was carried out with20% acetonitrile and 5 mM phosphate buffer at pH 6.5, using 0.01 mg/mLof purified peroxygenase (mature peroxygenase encoded by SEQ ID NO: 1)in a total reaction volume of 1 mL. The reaction was performed at roomtemperature for 5 minutes and stopped by adding 1 μL of catalase(Terminox Ultra 50L, Novozymes).

Samples were analyzed on an Agilent 1200 HPLC system equipped with aDiode Array Detector (Agilent, Santa Clara Calif., USA) and separated ona Gemini C6-Phenyl (110 Å, 2×150 mm, 3 μm) column from Phenomenex(Torrance Calif., USA) thermostated at 40° C. Two mobile phases wereused: (A) 0.1% formic acid, and (B) 0.1% formic acid in acetonitrile.

Separation was run using stepwise gradient starting with 40% B held for1 min, then increasing to 90% B within 4.5 min and being maintained at90% for 2 min with a constant flow rate of 0.4 mL/min.

Isobutylbenzene and its oxidation products,2-methyl-1-phenyl-1-propanol, isobutyrophenone,2-methyl-1-phenyl-2-propanol, were identified and quantified by externalcalibration using authentic standards, based on their retention times,UV absorption spectra (210 nm).

The peroxygenase oxidised isobutylbenzene (IBB) yielding three products,2-methyl-1-phenyl-1-propanol (MP1), isobutyrophenone (IBP), and2-methyl-1-phenyl-2-propanol (MP2), as shown in Table 8.

TABLE 8 Isobutylbenzene oxidation product yields. Product Yield (%) IBP4.7 MP1 35.1 MP2 5.5 Total product 45.3

Example 11 Oxidation of 1-Heptanol

Oxidation of 1 mM 1-heptanol with 1 mM H₂O₂ was carried out with 20%acetone and 10 mM phosphate buffer at pH 6, using 0.01 mg/mL of purifiedperoxygenase (mature peroxygenase encoded by SEQ ID NO: 1) in a totalreaction volume of 1 mL. Reaction was performed at room temperature for10 minutes and the sample was then inactivated by adding extractionsolvent ethyl acetate.

Sample was analyzed on an Agilent 7890A Gas Chromatograph equipped withAgilent 5975C series MSD system (Agilent, Santa Clara Calif., USA) and aZB-5HT (15×0.25 mm, 0.1 μm) column from Phenomenex (Torrance Calif.,USA). Helium was used as carrier gas at a constant flow rate of 2mL/min.

For analysis, 2 μL of ethyl acetate extract was injected into GC systemat 250° C. in the split mode (50:1). Separation was run usingtemperature program starting with 45° C. held for 1 min, then increasingto 50° C. at a rate of 5° C./min and holding for 1 min, then increasingto 200° C. at a rate of 30° C./min and holding for 0.5 min.

1-Heptanol and its possible oxidation products were identified andquantified by external calibration using authentic standards, based ontheir retention times and electron impact MS at 70 eV.

The peroxygenase oxidised 1-heptanol yielding a single product,heptaldehyde (10.6%).

Example 12 Cleavage of Benzyl Methyl Ether

Cleavage of 1 mM benzyl methyl ether with 1 mM H₂O₂ was carried out with20% or 30% acetonitrile, and 10 mM phosphate buffer at pH 6.0, using0.01 mg/mL of purified peroxygenase (mature peroxygenase encoded by SEQID NO: 1) in a total reaction volume of 1 mL. Reaction was performed atroom temperature for 25 minutes and stopped by adding 1 μL of catalase(Terminox Ultra 50L, Novozymes).

Sample was analyzed on an Agilent 1200 HPLC system equipped with a DiodeArray Detector (Agilent, Santa Clara Calif., USA) and separated on aGemini C6-Phenyl (110 Å, 2×150 mm, 3 μm) column from Phenomenex(Torrance Calif., USA) thermostated at 40° C. Two mobile phases wereused: (A) 0.1% formic acid, and (B) 0.1% formic acid in acetonitrile.

Separation was run using stepwise gradient starting with 10% B held for4 min, then increasing to 15% B within 0.5 min and being maintained for4 min, then increasing to 80% B within 7.5 min and being maintained for0.5 min, with a constant flow rate of 0.4 mL/min.

Benzyl methyl ether and its possible cleavage products, benzyl alcohol,benzaldehyde, and benzoic acid, were identified and quantified byexternal calibration using authentic standards, based on their retentiontimes, UV absorption spectra (210 nm, 230 nm, and 260 nm).

The peroxygenase oxidised benzyl methyl ether yielding two products,benzaldehyde and benzoic acid, as shown in Table 9.

TABLE 9 Yields of benzaldehyde and benzoic acid at 20% and 30%acetonitrile. Acetonitrile (%) Benzaldehyde (%) Benzoic acid (%) 20 22.07.2 30 9.6 1.1

Example 13 Oxidation of 2,3,6-Trimethyiphenol

Oxidations of 1 mM 2,3,6-trimethylphenol with 1 mM or 2 mM H₂O₂ werecarried out in 30% acetonitrile and 10 mM acetate (pH 3-5), phosphate(pH 6-7) or borate (pH 8) buffer at specified pH values (see Table 10)with 2 mM ascorbic acid, using 0.01 mg/mL of purified peroxygenase(mature peroxygenase encoded by SEQ ID NO: 1) in a total reaction volumeof 1 mL. Reactions were performed at room temperature for 15 minutes andstopped by adding 1 μL of catalase (Terminox Ultra 50L, Novozymes).

Samples were analyzed on an Agilent 1200 HPLC system equipped with aDiode Array Detector (Agilent, Santa Clara Calif., USA) and separated ona Luna C-18(2) (100 Å, 2.0×250 mm, 5 μm) column from Phenomenex(Torrance Calif., USA) thermostated at 40° C. Two mobile phases wereused: (A) 0.1% formic acid, and (B) 0.1% formic acid in acetonitrile.

Separations were run using stepwise gradient starting with 30% B andincreasing to 50% B within 0.5 min, then increasing to 80% B within 1.5min, and then again increasing to 95% B within 2 min, with a constantflow rate of 0.5 ml/min.

2,3,6-trimethylphenol and its oxidation product2,3,5-trimethyl-p-benzoquinone were identified and quantified byexternal calibration using authentic standards, based on their retentiontimes, UV absorption spectra (220 nm and 250 nm respectively).

Trimethylhydroquinone and other unidentified products were detected andquantified at 220 nm wavelength based on 2,3,6-trimethylphenolcalibration curve.

The peroxygenase oxidised 2,3,6-trimethylphenol yielding two products,2,3,5-trimethyl-p-benzoquinone (TMpBQ) and trimethylhydroquinone (TMHQ),as shown in Table 10.

TABLE 10 Comparison of 2,3,6-trimethylphenol oxidation product yields atvarious pH and hydrogen peroxide concentrations. H₂O₂ (mM) pH TMpBQ (%)TMHQ (%) 1 3 0 0 5 2 7 6 6 4 7 9 3 8 0 0 2 3 0 0 5 6 8 6 15 2 7 9 2 8 00

The invention claimed is:
 1. A method of producing a polypeptide having peroxygenase activity, comprising: (a) cultivating a microbial recombinant host cell under conditions conducive for production of the polypeptide, wherein the microbial recombinant host cell comprises a nucleic acid construct or expression vector comprising a polynucleotide encoding the polypeptide, the amino acid sequence of the polypeptide has at least 95% sequence identity to the sequence of amino acids 1 to 244 of SEQ ID NO: 2, and the polynucleotide is operably linked to one or more heterologous control sequences that direct the expression of the polypeptide in the recombinant host cell; and (b) recovering the polypeptide.
 2. The method of claim 1, wherein the amino acid sequence of the polypeptide has at least 96% sequence identity to the sequence of amino acids 1 to 244 of SEQ ID NO:
 2. 3. The method of claim 1, wherein the amino acid sequence of the polypeptide has at least 97% sequence identity to the sequence of amino acids 1 to 244 of SEQ ID NO:
 2. 4. The method of claim 1, wherein the amino acid sequence of the polypeptide has at least 98% sequence identity to the sequence of amino acids 1 to 244 of SEQ ID NO:
 2. 5. The method of claim 1, wherein the amino acid sequence of the polypeptide has at least 99% sequence identity to the sequence of amino acids 1 to 244 of SEQ ID NO:
 2. 6. The method of claim 1, wherein the polypeptide comprises the sequence of amino acids 1 to 244 of SEQ ID NO:
 2. 7. The method of claim 1, wherein the polypeptide is encoded by a polynucleotide that hybridizes under high stringency conditions with the full-length complement of (i) the sequence of nucleotides 52-1107 of SEQ ID NO: 1, wherein the high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours, and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C., and wherein the polypeptide has peroxygenase activity and has at least 95% sequence identity to the sequence of amino acids 1 to 244 of SEQ ID NO:
 2. 8. The method of claim 1, wherein the polypeptide is a variant of the sequence of amino acids 1 to 244 of SEQ ID NO: 2 comprising a substitution, deletion, and/or insertion at one or more positions, and wherein the polypeptide has peroxygenase activity and has at least 95% sequence identity to the sequence of amino acids 1 to 244 of SEQ ID NO:
 2. 9. The method of claim 1, wherein the polypeptide is a fragment of the sequence of amino acids 1 to 244 of SEQ ID NO: 2, and wherein the polypeptide has peroxygenase activity and has at least 95% sequence identity to the sequence of amino acids 1 to 244 of SEQ ID NO:
 2. 10. The method of claim 1, wherein the amino acid sequence of the polypeptide includes the motif: (SEQ ID NO: 7) E-H-D-[G,A]-S-[L,I]-S-R, or R-[G,A]-P-C-P-X-[M,L]-N-[S,T]-L,

wherein X is any amino acid (SEQ ID NO: 16).
 11. The method of claim 1, wherein the microbial recombinant host cell is a recombinant filamentous fungal host cell selected from the group consisting of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Mycefiophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, and Trichoderma.
 12. The method of claim 2, wherein the microbial recombinant host cell is a recombinant filamentous fungal host cell selected from the group consisting of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Mycefiophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, and Trichoderma.
 13. The method of claim 3, wherein the microbial recombinant host cell is a recombinant filamentous fungal host cell selected from the group consisting of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Mycefiophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, and Trichoderma.
 14. The method of claim 4, wherein the microbial recombinant host cell is a recombinant filamentous fungal host cell selected from the group consisting of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Mycefiophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, and Trichoderma.
 15. The method of claim 5, wherein the microbial recombinant host cell is a recombinant filamentous fungal host cell selected from the group consisting of Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Mycefiophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, and Trichoderma.
 16. The method of claim 11, wherein the recombinant filamentous fungal host cell is an Aspergillus or Trichoderma cell.
 17. The method of claim 12, wherein the recombinant filamentous fungal host cell is an Aspergillus or Trichoderma cell.
 18. The method of claim 13, wherein the recombinant filamentous fungal host cell is an Aspergillus or Trichoderma cell.
 19. The method of claim 14, wherein the recombinant filamentous fungal host cell is an Aspergillus or Trichoderma cell.
 20. The method of claim 15, wherein the recombinant filamentous fungal host cell is an Aspergillus or Trichoderma cell. 